1. Field of the Invention
The present invention relates to a method for identifying compounds having biological activity. More particularly, it relates to a method of identifying a ligand that binds to a biological substrate.
2. State of the Art
The function of a biological system may be modulated by the action of a ligand that binds to a biological substrate in the system, and so elicits a biological response. The ligand may mimic the action of an endogenous ligand, in which case it is referred to as an agonist, or it may block the action of an endogenous ligand, in which case it is referred to as an antagonist. Many biologically useful compounds, such as pharmaceuticals, pesticides and plant growth regulators, work as ligands in this way.
It is very important that a ligand binds selectively to a biological substrate, and so does not elicit a biological response in any other biological system. It is also important that the ligand binds strongly to the biological substrate, so that only a small concentration of the ligand is required to elicit the biological response.
Over the course of time, biological systems have evolved in which the endogenous ligands bind strongly and selectively to their respective biological substrates.
Methods for identifying exogenous ligands that bind strongly and selectively to a biological substrate have been developed, but these methods are time-consuming and require the making and testing of large numbers of candidates.
One method involves measuring the ability of candidates to elicit a biological response in a test biological system, and then evaluating the selectivity of an active candidate by measuring its ability not to elicit a biological response in another biological system, or in a whole organism.
Another method involves measuring the ability of a candidate to displace a known ligand, such as the endogenous ligand or an agonist. In this method, the known ligand is used as a probe. It may be radiolabelled, so as to facilitate measuring the amount of the known ligand that is bound or unbound to the biological substrate in the presence and absence of the candidate.
Another method for identifying a ligand is disclosed in U.S. Pat. No. 5,698,401 (Fesik). This method makes use of a 15N labeled biological substrate and an NMR technique. In the method, a 15N/1H two dimensional correlation spectrum of a biological substrate in the presence of a candidate is compared with a 15N/1H two dimensional spectrum in the absence of a candidate. By comparing the spectra obtained at different concentrations of the candidate, a dissociation constant, Kd, between the biological substrate and the candidate may be determined.
International patent application, Publication No. WO 00/00823 discloses a method of identifying a ligand in which a biological substrate that contains a chemically reactive group (or has been modified to contain a chemically reactive group) is exposed to a test compound capable of reacting with the chemically modified group. The method assumes that a test compound that possesses binding affinity for the biological substrate will tend to react more favorably with the chemically reactive group that a test compound that does not possess binding affinity.
Biological substrates may contain more than one site to which a ligand may bind.
Recently, interest has been growing in ligands that have the ability to bind to more than one binding site on a biological substrate (Borman, S., Oct. 9, 2000, CandEN, 48-53). Such ligands are referred to as multibinding or multivalent compounds or ligands. These multibinding ligands comprise two or more binding regions connected by one or more linkers. The linkers constrain the relative positions and orientations of the binding regions, so that each binding region can bind at the same time to the respective binding sites on the biological substrate. The biological substrate may be made up of a single unit or a cluster of two or more units, in which case a multivalent compound may bind simultaneously to binding sites on two or more units.
Multibinding ligands bind more strongly and selectively to biological substrates than monovalent ligands. This is because the presence of multiple binding regions means that it is less likely that the whole ligand will become unbound, and because it is less likely that two different biological substrates will possess the same multiple binding sites in the same relative positions.
WO 99/42476 and WO 99/64032 disclose an iterative method for identifying multibinding ligands in which candidates are prepared by connecting a diversity of fragments of known ligands through a diversity of linkers, then determining whether the candidates possess multibinding properties. A successful candidate will contain fragments of known ligands that are capable of binding to different binding sites on the biological substrate and are presented by the linker in such a way that they may bind simultaneously to their respective binding sites.
Application of the method disclosed in WO 99/42476 and WO 99/64032 has enabled the rapid identification of highly potent and selective ligands. However, there remains a need for other methods.
A method has now been devised for identifying ligands that makes use of the binding affinity of a ligand for one binding site on a biological substrate to identify a ligand that binds to another.
According to one aspect, therefore, the present invention provides a method of identifying a ligand for a second binding site on a biological substrate, which comprises determining the effect of a candidate compound on the binding of a probe compound which binds to a first binding site on the biological substrate in the presence of a first multibinding compound which binds to the first and second binding sites on the biological substrate.
A successful candidate compound will compete with the first multibinding compound for binding to the second binding site, and so reduce the binding of the first multibinding compound to the biological substrate. This reduced binding of the first multibinding compound may be detected by the increased binding of the probe compound.
Thus, in another aspect, the present invention provides a method of determining whether a candidate compound is a ligand for a second binding site of a biological substrate, the method comprising:
(a) contacting a biological substrate comprising a first binding site and a second binding site with a candidate compound in the presence of (i) a probe compound which binds to the first binding site of the biological substrate and (ii) a first multibinding compound which binds to the first binding site and the second binding site of the biological substrate;
(b) determining the amount of probe compound that binds to the first binding site of the biological substrate in step (a);
(c) comparing the amount of probe compound determined in step (b) with a pre-determined amount of probe compound that binds to the first binding site when the biological substrate is contacted with the probe compound and the first multibinding binding compound in the absence of the candidate compound.
If the candidate compound is a ligand for the second binding site of the biological substrate, more of the probe compound will bind to the first binding site of the biological substrate in the presence of the candidate compound than in its absence.
Additionally, the relative binding affinities of candidate compounds can be determined using the method of this invention by comparing the relative amounts of the probe compound that bind to the biological substrate in the presence of each of the candidate compounds. Therefore, in yet another aspect, the present invention provides a method of determining the relative binding affinity of a plurality of candidate compounds for a second binding site of a biological substrate, the method comprising:
(a) contacting a biological substrate comprising a first binding site and a second binding site with each of a plurality of candidate compounds in the presence of (i) a probe compound which binds to the first binding site of the biological substrate and (ii) a first multibinding compound which binds to the first binding site and the second binding site of the biological substrate;
(b) determining the amount of probe compound that binds to the first binding site of the biological substrate for each of the candidate compounds in step (a);
(c) comparing the amount of probe compound determined in step (b) for each of the candidate compounds.
Since the amount of probe compound that binds to the first binding site of the biological substrate in the presence of each candidate compound is related to that candidate compound""s affinity for the second binding site, the relative affinity of each candidate compound for the second binding site can be determined.